| BackgroundCoronary no reflow (NR) phenomenon is one of the most serious clinicalchallenges that influence the prognosis of patients with acute coronary syndrome.Coronary no reflow phenomenon of varying degrees widely exists in patientsundergoing coronary artery recanalization therapy with type2diabetes mellitussignificantly increasing the severity of it. Lots of present studies showed that thesignificant decline of adiponectin (APN) secreted by adipocyte, led tohypoadiponectinemia, which could greatly increase the risk for coronary heart disease.As a consequence, hypoadiponectinemia is an independent risk factor for coronaryheart disease. Meanwhile, circulating adiponectin concentration in patients with type2diabetes mellitus is observably dropped. Related studies showed that decreasedsecretion of adiponectin, which participated in the development of insulin resistanceas a result of obesity, could increase myocardial ischemia/reperfusion injury due todecreased cardiovascular protective effect of adiponectin. No reflow, which is theresult of ischemia/reperfusion injury appeared in coronary microcirculation, is muchmore serious in type2diabetes mellitus. It is still unknown whether or not attenuatedcardiovascular protection of adiponectin contributes to the aggravated no reflowinjury in type2diabetes mellitus. In our study, we shall firstly demonstrate ourhypothesis that whether hypoadiponectinemia could lead to aggravated coronaryno-reflow injury in type2diabetes mellitus at different levels like organs, tissues andcells progressively. Furthermore, we shall explore the protective effect of adiponectinon coronary microcirculation to deepen the understanding of mechanisms ofno-reflow phenomenon and to provide experimental evidences for better clinicaltreatment strategies of coronary no-reflow.Methods1. Model preparation of coronary no reflow in type2diabetic rats1) Type2diabetic rats induced by continuous high-sugar and high-fat feeding.75healthy male Sprague-Dawley (SD) rats of6-week-old were selected in ourexperiments, and they were divided into two groups--10rats in control group which were fed with normal diet, and65rats in high-sugar and high-fat group which werefed with diet containing60%high fat and sugar. All rats were fed continuously inbarrier environment for32weeks. Body weight, fasting glucose, fasting insulin andserum adiponectin levels were monitored regularly. Eight weeks later, intraperitonealinjections of glucose tolerance test（IPGTT）was performed every4weeks. Andabnormal IPGTT was selected as measurement indicators for successful model oftype2diabetes.2) Model preparation of coronary no reflow in type2diabetic rats.Successfully fed type2diabetic rats and normal rats were used in followingexperiments:①Shamoperation group (Sham group)(n=5):0.5ml of saline solution was injected into rats via tail vein at the end of31stweek.②Normal control group (Normal group)(n=5):0.5ml of saline solution was injected into rats via tail vein at the end of31stweek.③diabetic rats group (DM group)(n=7):0.5ml of saline solution was injected into rats via tail vein at the end of31stweek.④diabetic rats+globular adiponectin intervention group (DM+gAd group)(n=6):Globular adiponectin withdose of20μg/kg was injected into rats via the tail veinat the end of31st week.In each group, myocardial ischemia/reperfusion injury model was established byligating left anterior descending coronary artery for1hour and a half for ische mia and12hours for reperfusion. No reflow injury areas were measured using Evans blueand Thioflavin S staining methods. Left ventricular pressure was determined toevaluate cardiac function of rats. Serum adiponectin (APN), endothelin-1(ET-1),intercellular adhesion molecule-1(ICAM-1) and vascular endothelial adhesionmolecule-1(VCAM-1) levels were determinated simultaneously to assess vascularendothelial injury. 2. In vitro vascular rings experiment of type2diabetic ratsVascular rings of thoracic aorta and coronary artery branches of3rdlevel wereisolated from type2diabetic rats. Experimental groups are as follows:①Normal control group (Normal group)(n=5);②Diabetic rats group (DM group)(n=5).③Diabetic rats+globular adiponectin incubation group (DM+gAd group)(n=6).In vitro blood vessels were incubated with vehicle (Normal group, DM group) orgAd (DM+gAd group) with dose of2μg/ml in a cell culture incubator. After4h ofincubation, endothelial dependent vasomotor function tests were performed usingDMT tension apparatus with PowerLab data acquisition system by microvascular ringtechnique.3. Hypoxia/reoxygenation (H/R) model of human umbilical vein endothelial cells(HUVECs) after high glucose stimulation.Primary endothelial cells were isolated and cultured from human umbilical vein.Hypoxia/reoxygenation model is established after high glucose stimulation.Experimental groups are as follows:①Vehicle group；②hypoxia3h/reoxygenation1h group (H/R group);③10mmol/L high glucose stimulation group (G group);④10mmol/L high glucose stimulation+hypoxia3h/reoxygenation1h group(G+H/R group);⑤10mmol/L high glucose stimulation+hypoxia3h/reoxygenation1h+gAd group(G+H/R+gAd group).Group3rd to5th were incubated with10mmol/L high glucose for48h. The5thgroup was adminitrated with adiponectin for24h, with final concentration in cellculture medium at2μg/ml. Neither vehicle group nor H/R group had drug treatment.ET-1, ICAM-1, VCAM-1and E-selectin in supernates of each group of HUVECswere detected.Results1. Models of coronary no reflow in type2diabetic rats 1) Serum adiponectin levels of diabetic rats increased firstly and then continued toreduce in the process of long-term high-sugar and high-fat feeding, whosevariations was consistent with insulin changes but was slower than than insulin.2) ET-1, ICAM-1, VCAM-1in DM group and DM+gAd group were significantlyincreased in the32th weekend.After administration of gAd, ET-1wassignificantly reduced (108.6.13.±8.428vs.132.10±7.213，P<0.05).3) No reflow injury in type2diabetic rats was increased after ischemia/reperfusion(ANR/AAR：48.64±2.93%vs.35.48±4.31%, P0.05), cardiac function showedobvious impairation with increased serum levels of adhesion moliecules(ICAM-1, VCAM-1).4) After administration of gAd, no reflow injury and cardiac function afterischemia/reperfusion injury in type2diabetic rats improved significantly withdecreased serum adhesion molecules (ICAM-1, VCAM-1) levels.5) APN protein content in areas of no reflow was significantly lower than that inareas of reperfusion within ischemic areas in type2diabetic rats2. In vitro vascular ring experiment in type2diabetic ratsThoracic aorta endothelium dependent vasodilatation function: Severeendothelial dysfunction was observed in aortic segments of type2diabetic rats(70±3.57%vs.93±6.12%in control, P0.05), while administration with gAdsignificantly improved endothelial function.3. Hypoxia/reoxygenation model of HUVECs after high glucose stimulationCompared with blank control group (Vehicle), concentrations of ET-1, E-selectin,ICAM-1and VCAM-1in supernatants of H/R group, G group and G+H/R groupincreased significantly; compared with G+H/R group, concentrations of ET-1,E-selectin, ICAM-1and VCAM-1in supernatants of G+H/R+gAd group decreasedobviously after pretreatment of gAd.ConclusionsAggravated injury of coronary no reflow after myocardial ischemia/reperfusionin type2diabetes mellitus is associated with lower serum adiponectin levels, andexogenous administration of adiponectin could effectively alleviate coronary no reflow injury in type2diabetic rats and improve cardiac function as well. It issupposed that adiponectin plays the significant role of microcirculation protection byreducing adhesion molecules secretion by endothelial cells. |
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